Intracellular metabolism is a complex network of intersecting reactions in all living things. In Gram-positive bacteria, the CodY protein is a major integrator of diverse metabolic pathways and regulatory schemes, helping the cell to turn many pathways on or off in response to the intracellular pools of four metabolites, the three branched-chain amino acids (BCAAs; isoleucine, leucine and valine) and GTP. In pathogenic Gram-positive bacteria, CodY is also a major regulator of virulence genes. Depending on the organism and the role of a specific virulence gene product, CodY, may act as either a negative or a positive regulator and may either inhibit or stimulate pathogenesis. This property of CodY links pathogenesis in an important way to the physiological state of the cell by tying the bacterium's decision to cause damage to the host to the pools of just a few key metabolites. Not all CodY target genes, however, are equally sensitive to CodY-mediated regulation. That is, variations in the intracellular pools of the BCAAs and GTP due to variations in nutrient availability create situations in which different fractions of the total population of CodY molecules are active. As a result, some genes (e.g., those that have very high affinity targets for CodY) are fully repressed under conditions in which other genes (e.g., those with low affinity targets) are hardly repressed at all. Thus, CodY-regulated genes fall within a regulatory spectrum or hierarchy. A major goal of the proposed project is to determine where each CodY-regulated gene in the model organism, Bacillus subtilis, falls within this spectrum, to couple the positioning of genes to the intracellular pools of the CodY effector molecules, to determine the molecular mechanisms that are responsible for establishing the spectrum of gene regulation, and to identify the metabolic pathways that are induced or repressed at different levels of nutrient availability. This analysis s expected to provide novel insight into how the cell prioritizes the usage of specific metabolic pathways in response to general nutrient limitation. Analogous studies with two important human pathogens, Staphylococcus aureus and Clostridium difficile, will reveal where within the CodY hierarchy various virulence genes fall. CodY is a major repressor of virulence in both species. The results will give an unprecedented view of the extent of specific metabolic limitation that causes a bacterium to turn from a commensal life style to a pathogenic life style. Moreover, for a complex pathogen, such as S. aureus, that expresses dozens of virulence factors that cause different extents of damage to the host, it will be possible to learn to what extent metabolite pools have to drop before the most damaging factors are induced. PUBLIC HEALTH RELEVANCE: Bacteria have a remarkable ability to adapt to many different environmental conditions and stresses. This ability greatly influences both their choice of habitats and the conditions under which they cause human disease. This proposal seeks to reveal a principal mechanism by which bacteria couple the expression of pathogenesis genes to the accumulation of specific intracellular compounds. Knowing which compounds affect pathogenesis is expected to lead to the development of novel anti-infectives.